Method and algorithm for quantifying polynucleotides

Abstract

Machine executable method of analyzing growth curve data to identify the transition from a baseline phase into a growth phase. Applications of the method include analysis of results from time-dependent monitoring of amplicon synthesis in a nucleic acid amplification reaction to quantify a starting amount of a nucleic acid template in a test sample. The method advantageously simplifies the quantitation by circumventing the need to establish thresholds used for calculating initiation of the growth phase, to calculate derivatives, or to perform linear regression analysis.

1. A method for determining the amount of a nucleic acid in a test sample, comprising the steps of:
 amplifying a predetermined locus of the nucleic acid in an in vitro amplification reaction to create a nucleic acid amplification product; 
 determining a value proportional to the amount of the nucleic acid amplification product present at different times during the in vitro amplification reaction, whereby there is created a collection of time-dependent values that collectively define a growth curve; 
 performing a vector analysis on at least a portion of said growth curve to identify a time-dependent feature indicating a transition from the baseline phase into the growth phase of said growth curve, said vector analysis comprising the steps of,
 (a) establishing a plurality of pairs of first and second vectors at different points on a time dimension of the growth curve using said collection of time-dependent values,
 wherein each vector of a single pair of vectors among said plurality has the same origin, and 
 wherein the head of the first vector and the head of the second vector of said single pair of vectors among said plurality are each positioned at different points on the growth curve, 
 
 (b) calculating for each pair of vectors the value of an angle therebetween, thereby resulting in a plurality of calculated angles, with each point on the time dimension of the growth curve being associated with a different one of said plurality of calculated angles, 
 (c) comparing the plurality of calculated angles with each other to identify a characteristic angle, and 
 (d) identifying said time-dependent feature of said growth curve as the point on the time dimension of the growth curve that is associated with the characteristic angle, said characteristic angle being either the maximum angle among the plurality of calculated angles or the minimum angle among the plurality of calculated angles; 
 
 comparing the time-dependent feature identified in step (d) with a standard calibration curve; and 
 determining from said time-dependent feature of said growth curve and said standard calibration curve the amount of the nucleic acid in the test sample. 
 
     
     
       2. The method of  claim 1 , wherein said characteristic angle is the minimum angle among the plurality of calculated angles. 
     
     
       3. The method of  claim 1 , wherein said characteristic angle is the maximum angle among the plurality of calculated angles. 
     
     
       4. The method of  claim 1 , wherein each vector in said single pair of vectors has a different magnitude in said time dimension of said growth curve, said vectors being directionally similar vectors, and wherein said characteristic angle is the maximum angle among the plurality of calculated angles. 
     
     
       5. The method of  claim 1 , wherein each vector in said single pair of vectors has a different magnitude in said time dimension of said growth curve, said vectors being directionally opposed vectors, and wherein said characteristic angle is the minimum angle among the plurality of calculated angles. 
     
     
       6. The method of  claim 1 , wherein each vector in said single pair of vectors has the same magnitude in said time dimension of said growth curve, said vectors being directionally opposed vectors, and wherein said characteristic angle is the minimum angle among the plurality of calculated angles. 
     
     
       7. The method of  claim 1 , further comprising a processing step before the step for performing the vector analysis, said processing step comprising processing said collection of time-dependent values of the first determining step by a smoothing function to result in smoothed numerical data, and wherein the collection of time-dependent values used in step (a) of the vector analysis is the smoothed numerical data resulting from the processing step. 
     
     
       8. The method of  claim 7 , wherein the smoothing function in the processing step is selected from the group consisting of a moving average smoothing function, and a curve-fitting smoothing function. 
     
     
       9. The method of  claim 7 , wherein each vector in said single pair of vectors has a different magnitude in said time dimension of said growth curve, said vectors being directionally similar vectors, and wherein said characteristic angle is the maximum angle among the plurality of calculated angles. 
     
     
       10. The method of  claim 7 , wherein each vector in said single pair of vectors has a different magnitude in said time dimension of said growth curve, said vectors being directionally opposed vectors, and wherein said characteristic angle is the minimum angle among the plurality of calculated angles. 
     
     
       11. The method of  claim 7 , wherein each vector in said single pair of vectors has the same magnitude in said time dimension of said growth curve, said vectors being directionally opposed vectors, and wherein said characteristic angle is the minimum angle among the plurality of calculated angles. 
     
     
       12. The method of  claim 1 , wherein said in vitro amplification reaction in the amplifying step is an isothermal in vitro amplification reaction. 
     
     
       13. The method of  claim 12 , wherein the value proportional to the amount of the nucleic acid amplification product in the first determining step is a fluorescence value. 
     
     
       14. The method of  claim 1 , wherein the step for performing the vector analysis is automated by a computer. 
     
     
       15. The method of  claim 1 , wherein the determined amount of said nucleic acid in the test sample is recorded in tangible form. 
     
     
       16. The method of  claim 1 , wherein the steps for performing a vector analysis, comparing the time dependent feature, and determining from said time-dependent feature, are automated by a computer.